Amyotrophic Lateral Sclerosis (ALS) is epidemiologically classified into sporadic (90%-95%) and familial (5%-10%) forms (Rosen et al., 1993). Twenty percent of the familial forms (fALS) are caused by mutations in the Superoxide Dismutase 1 (SOD1) gene. The function of the SOD1 metalloenzyme is to convert superoxide, a toxic by-product of mitochondrial oxidative phosphorylation, to molecular oxygen or hydrogen peroxide. Mutant SOD1 possesses a neurotoxic property (toxic gain of function) that is responsible for the pathogenic mechanism of the disease. Indeed, transgenic mice overexpressing mutant forms of the human SOD1 gene (for example SOD1G93A mice) recapitulate most pathological features of ALS and are widely used in ALS preclinical studies (Gurney et al., 1994). Decreasing the accumulation of SOD1 has thus arisen as a logical strategy to treat SOD1-linked forms of fALS. Attractive molecular approaches have been developed to downregulate almost any gene in the central nervous system (CNS), mainly based on the use of antisense oligonucleotides (AONs) (Crooke, 2004) or RNA interference with either siRNA (Dorn et al., 2004) or synthetic microRNA (Boudreau et al., 2011).
Suppression of mutant SOD1 expression using siRNA has first proved significant therapeutic efficiency in SOD1-linked ALS mice. Raoul et al. showed that intraspinal injection of lentiviral vectors encoding short hairpin RNAs (shRNAs) to human SOD1 delayed disease onset and progression in SOD1G93A mice (Raoul et al., 2005). Independently, Ralph et al., demonstrated that intramuscular injections of lentivirus mediating the expression of RNAi to the human SOD1, prevented neurodegeneration and extended survival in the same ALS mouse model, leading to a maximal 77% lifespan increase (Ralph et al., 2005).
Continuous infusion of an AON inducing enzyme-mediated decay into the brain ventricles has also been reported to allow efficient and widespread reduction of both SOD1 mRNA and protein levels throughout the brain and the spinal cord, significantly slowing disease progression in a rat model of ALS caused by the SOD1G93A mutation (Smith et al., 2006).
However, this method necessitated surgically implantation of a catheter through the skull, connected to an osmotic pump and its therapeutic efficacy was limited (9.1% extension survival with a treatment beginning at 65 days of age) (Smith et al., 2006). Based on this discovery, a multicenter clinical trial of AONs infusion into ALS patient's cerebrospinal fluid (CSF) was initiated by Isis Pharmaceuticals, showing the feasibility and the lack of adverse effects of the treatment (Miller et al., 2013). More recently, steric blocking AONs were also used to promote aberrant exon-skipping (and generation of premature stop codon containing mRNAs), as an alternative method to decrease mouse Sod1 levels in the CNS of wild type mice (Ward et al., 2014). However, the intracerebroventricular (ICV) injection of 2′-MOE AONs targeting mouse Sod1 pre-mRNA caused only a weak skipping of Sod1 exon 2 and exon 3 in the brain and spinal cord, leading to 25-50% reduction of Sod1 levels, similarly to the level achieved with the same dose of the previously used RNase H-dependent 2′-MOE gapmer AONs in SOD1G93A rats (Smith et al., 2006). From these results, lifespan improvement would have been expected to be, at most, equivalent to that obtained with enzyme-mediated strategies such as RNAi or gapmer strategies.
In addition, the immediate challenge facing fALS therapies based on SOD1 suppression is the widespread delivery of the silencing instructions to all affected cells. In 2007, we discovered that, despite the blood-brain-barrier, systemic delivery of self-complementary adeno-associated virus vectors of serotype 9 (scAAV9) allowed transduction of both CNS and peripheral cells in mice and cats, including in the cell types suspected to be involved in ALS (neurons, astrocytes, and muscle cells) (Duque et al., 2009) (EP2212424). More recently, the rh10 serotype (AAV10) was also found efficient for systemic transduction of CNS and peripheral tissues after IV injection in mice and marmosets (Hu et al., 2010; Yang et al., 2014; Zhang et al., 2011).
Recently, the efficiency of AAV-based gene therapy strategies for ALS has been demonstrated in two studies using RNA interference to reduce SOD1 levels. Foust et al. first obtained a 38% of survival extent in ALS mice following intravenous (IV) injection of neonates with an AAV9-shRNA targeting SOD1 (Foust et al., 2013). Furthermore, intrathecal (IT) injection of an AAV10-shRNA-SOD1 in post-symptomatic 55-days old SOD1 mice resulted in 22% of increased survival in ALS mice (Wang et al., 2013).
In view of the limited therapeutic achievements reported in these previous studies, technology improvements for ALS biotherapy are still needed.